The advent of recombinant DNA technology has made possible the large scale production of proteins by the insertion of heterologous, protein-encoding genes into microorganisms such as bacteria and expression of the genes within the host microorganisms. In this manner, a large variety of proteins, including some which can only be obtained in minute quantities from natural sources, can be economically produced in unlimited quantities.
Unfortunately, proteins which are native to eukaryotic cells may not undergo post-translational processing in microbial hosts to yield biologically active forms of the desired proteins. Thus, for example, eukaryotic proteins containing multiple cysteine residues may not form the correct disulfide linkages necessary for biological activity when they are expressed in microbial hosts. Not only may the eukaryotic protein fold improperly within the intracellular environment of the host, but also the individual molecules may form biologically inactive aggregates or oligomers as the result of the formation of intermolecular disulfide bonds or other types of intermolecular bonding.
As the result of one or more of these phenomena--improper folding, incorrect disulfide bond formation and non-covalent or covalent oligomerization--many proteins that are produced by the expression of heterologous genes in microbial hosts are not recovered from the host cells in the form of soluble, biologically active protein. Rather, upon lysis of the cells, the heterologous proteins are found in the form of insoluble "inclusion bodies," also sometimes referred to as "refractile bodies." In order to produce useful proteins, a means must be provided whereby the inclusion body proteins can be converted into a monomeric, biologically active form in which they are soluble in biological fluids.
In addition to converting the inclusion body proteins into soluble, monomeric, biologically active forms, it is necessary at some point in the recovery process to purify the protein in order to remove bacterial impurities including endotoxins, other bacterial proteins and contaminating substances derived from the bacterial host and/or the fermentation medium. This is usually done by subjecting the protein to some of the various chromatographic purification procedures such as ion-exchange chromatography.
PCT Application No. GB 83/00152 discloses methods for recovering and activating the milk-clotting enzyme chymosin, beginning with inclusion bodies produced in E. coli which contain the enzyme in its zymogenic form. The methods involve dissolving the inclusion body protein in denaturants such as urea, guanidine hydrochloride or alkali solution, renaturing the protein by removing or diluting the denaturant and reducing the pH of the solutions to induce autocatalytic cleavage of the zymogen to the mature form of the protein.
Solubility and folding characteristics vary considerably between different proteins, since both are highly dependent on the primary structure, i.e., amino acid sequence, of the protein. It has been the experience of the prior art that animal growth hormones are particularly difficult proteins to recover in soluble, monomeric, biologically active form. Thus, for example, it is said in U.S. Pat. No. 4,512,922 that, for proteins such as growth hormones, dissolution of the inclusion body protein in a strong denaturant followed by dilution of the denaturant with aqueous buffer almost invariably results in reprecipitation of the protein. Even if reprecipitation does not occur, expected levels of activity are said not to be shown. As a solution to this problem, there is disclosed a method for purifying growth hormone in which the inclusion body proteins are solubilized in a strong denaturant; the strong denaturant is replaced by a weaker denaturant; and the weaker denaturant is subsequently removed to renature the protein.
We have found that a two-stage renaturation process, such as that disclosed in U.S. Pat. No. 4,512,922, entails a number of problems. The yields of soluble, biologically active growth hormone obtainable are not particularly good. Moreover, the method yields varying results depending on the species of growth hormone involved. For example, using 8 M guanidine hydrochloride as a strong denaturant and 3.5 M urea as the weaker denaturant, we have found that yields of soluble, biologically active porcine growth hormone were only on the order of about 1% or less. While bovine growth hormone yields were somewhat higher, on the order of about 5%, these were still only marginal from a commercial point of view. Moreover, problems arose in the purification of the proteins recovered by this process. When the proteins recovered in this manner were loaded onto an ion-exchange column for purification, large quantities of soluble protein aggregates bound to the column, causing it to become fouled and obstructed within a relatively short period of time. This was true even when the column purification was carried out under reducing conditions in an attempt to eliminate aggregates. The use of the two-stage renaturation process is also problematical from a commercial production standpoint inasmuch as it entails numerous processing steps and expensive reagents.
We have also attempted to recover growth hormones from inclusion bodies by solubilizing the inclusion body proteins in 8 M urea and subsequently renaturing the protein in a single step by dialysing the solution against denaturant-free buffer to remove the urea. Yields of recovered monomeric growth hormone were very poor, that is, on the order of 1% or less.
It is an object of this invention to provide an efficient method for recovering microbially produced growth hormone in a soluble, monomeric, biologically active form.
It is a further object of the invention to provide a method for recovering and purifying microbially produced growth hormone using a chromatographic purification column whereby the purification column does not become plugged and obstructed within a short period of time.
Other objects and advantages of the invention will be readily apparent from the description of the invention which follows.